Activation of immune system cells via ligation of their cell surface CD40 molecules can induce both T cell- dependent and independent antitumor effects in tumor-bearing mice. We have recently shown that CD40 and Toll-like receptor-9 (TLR9) ligation by the combination of two immunomodulators, anti-CD40 monoclonal antibody (1CD40) and CpG, induces synergistic activation of macrophages and results in potent antitumor responses even in mice lacking T, B and cytolytic NK cells. As macrophages can be relatively resistant to chemotherapy, we hypothesize that in vivo macrophage activation with 1CD40 plus CpG therapy will augment the antitumor effect of prior chemotherapy in tumor-bearing hosts. Recent preliminary data support this hypothesis. We also hypothesize that targeting macrophages to the tumor microenvironment using a fusion protein consisting of a tumor reactive monoclonal antibody linked to the CCL16 chemokine will further enhance their antitumor effects in mice with established tumors. We also hypothesize that strategies known to interfere with tumor induced immune-suppression will enable even greater macrophage mediated antitumor activity following 1CD40 and CpG treatment. The purpose of this project is to characterize the antitumor mechanisms and therapeutic utility of 1CD40+CpG-activated macrophages. Specifically we will accomplish this objective through the following 4 aims, which include in vitro and in vivo analyses of murine macrophages and their effects on murine tumors, particularly the B16 melanoma: 1. Utilize 1CD40+CpG to overcome tumor-induced suppression of MF. 2. Determine how pretreatment with specific chemotherapy agents synergizes with the antitumor effects of MF activated with 1CD40+CpG. 3. Determine if localizing of 1CD40+CpG-MF in the tumor using chTNT-3/LEC FP will result in better antitumor effects. 4. Evaluate preclinical strategies of combinatory 1CD40+CpG therapy. This research will determine how to augment antitumor reactivity by macrophages activated with 1CD40 and CpG by interfering with tumor induced immune suppression in vivo. It will then identify combined regimens that further enhance these antitumor effects in mice with established tumors. As both murine and human macrophages can be activated through CD40 and TLR9, these basic and preclinical studies should provide a framework for future clinical trials utilizing this novel strategy of combining chemotherapy with macrophage- activating and localizing modalities. PUBLIC HEALH RELEVANCE: Macrophages are important derivatives of white blood cells that are critical in our defense against infections and in activating protective immune responses. This proposal is based on the concept that macrophages can also be activated to mediate direct anticancer effects; we hypothesize that proper activation of macrophages in patients with cancer has great therapeutic potential, especially in combination with other anti-cancer therapies, including conventional chemotherapy. The overall goal of this proposal is to characterize and further develop this novel strategy for macrophage activation in cancerous mice, where beneficial anti-cancer effects have already been shown, in order to determine how best to use this as a future strategy for treating human cancers.